Evaluation of alternative killing agents for Aedes … · Evaluation of alternative killing agents for Aedes aegypti (Diptera: Culicidae) in the Gravid Aedes Trap (GAT) Journal: Journal

Evaluation of alternative killing agents for Aedes aegypti

(Diptera: Culicidae) in the Gravid Aedes Trap (GAT)

Journal: Journal of Medical Entomology

Manuscript ID JME-2015-0351.R1

Manuscript Type: Research Article

Date Submitted by the Author: n/a

Complete List of Authors: Heringer, Laila; Laboratório de Ecologia Química de Insetos Vetores (LabEQ), Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil Johnson, Brian; James Cook University, College of Public Health, Medical and Veterinary Sciences; James Cook University, Australian Institute of Tropical Health and Medicine Fikrig, Kara; James Cook University, College of Public Health, Medical and Veterinary Sciences; Yale University, Yale School of Public Health Townsend, Michael; James Cook University, College of Public Health, Medical and Veterinary Sciences; James Cook University, Australian Institute of Tropical Health and Medicine Barrera, Roberto; Centers for Disease Control and Prevention., Entomology and Ecology Activity, Dengue Branch; Centers for Disease Control and Prevention., Entomology and Ecology Activity, Dengue Branch Eiras, Álvaro; Laboratório de Ecologia Química de Insetos Vetores (LabEQ), Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil Ritchie, Scott; James Cook University, College of Public Health, Medical and Veterinary Sciences; James Cook University, Australian Institute of Tropical Health and Medicine

<b>Please choose a section from the list</b>:

Vector Control, Pest Management, Resistance, Repellents

Field Keywords: Dengue, Mosquito Control, Insecticide Resistance

Organism Keywords: Aedes aegypti

https://mc.manuscriptcentral.com/medent

Manuscripts submitted to Journal of Medical Entomology

1

Heringer et al: Alternative killing agents for Gravid Aedes Trap Journal of Medical Entomology Research Article

Title: Evaluation of alternative killing agents for Aedes aegypti (Diptera: Culicidae) in 1

the Gravid Aedes Trap (GAT) 2

3

Author's names: Laila Heringer1,2, Brian Johnson2,3, Kara Fikrig2,4, Bruna A. Oliveira1, 4

Richard D. Silva1, Michael Townsend2,3, Roberto Barrera5, Álvaro Eduardo Eiras1, Scott 5

A. Ritchie2,3,* 6

7

Institutional affiliations: 8

1Laboratório de Ecologia Química de Insetos Vetores (LabEQ), Departamento de 9

Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 10

Brasil. 11

2College of Public Health, Medical and Veterinary Sciences, James Cook University, 12

PO Box 6811 Cairns Queensland 4870 Australia. 13

3Australian Institute of Tropical Health and Medicine, James Cook University, PO Box 14

6811 Cairns Queensland 4870 Australia. 15

4Yale School of Public Health, Yale University, 60 College Street, P.O. Box 208034, 16

New Haven, CT 06520 United States. 17

5Entomology and Ecology Activity, Dengue Branch, Centers for Disease Control and 18

Prevention,1324 Calle Cañada, San Juan, Puerto Rico 00920 19

Page 1 of 28



2

*Corresponding Author: Scott A. Ritchie, College of Public Health, Medical and 20

Veterinary Sciences, James Cook University, Building E4, McGregor Rd, Smithfield, 21

4878 QLD Australia, Phone: +61 7 4232 1202, Fax: +61 7 4232 1251, Email: 22

[email protected]

24

Page 2 of 28



3

Abstract: The Gravid Aedes Trap (GAT) uses visual and olfactory cues to attract gravid 25

Aedes aegypti that are then captured when knocked down by a residual pyrethroid 26

surface spray. However, the use of surface sprays can be compromised by poor 27

availability of the spray and pesticide resistance in the target mosquito. We investigated 28

several ‘alternative’ insecticide and insecticide-free killing agents for use in the GAT. 29

This included long-lasting insecticide-impregnated nets (LLINs), vapor active SPs 30

(metofluthrin), canola oil and two types of dry adhesive sticky card. During bench top 31

assays LLINs, metofluthrin, and dry sticky cards had 24 hr knockdown (KD) 32

percentages >80% (91.2±7.2%, 84.2±6.8%, and 83.4±6.1%, respectively), whereas the 33

24 hr KD for canola oil was 70±7.7%, which improved to 90.0±3.7% over 48 hr. 34

Importantly, there were no signigicant differences in the number of Ae. aegypti 35

collected per week or the number of traps positive for Ae. aegypti between the sticky 36

card and canola oil treatments compared to the surface spray and LLIN treatments in 37

semi-field and field trials. These results demonstrate that the use of inexpensive and 38

widely available insecticide-free agents such of those described in this study are 39

effective alternatives to pyrethoids in regions with insecticide resistant populations. The 40

use of such environmentally friendly insecticide-free alternatives will also be attractive 41

in areas where there is substantial resistance to insecticide use due to environmental and 42

public health concerns. 43

44

Key words: Aedes aegypti, dengue, Zika, mosquito trap, entomological surveillance 45

Sponsorships: CAPES, CNPq and FAPEMIG. National Health and Medical Research 46

Council Senior Research Fellowship 1044698 (www.nhmrc.gov.au). 47

48

49

Page 3 of 28



4

Introduction 50

Aedes (Stegomyia) aegypti (L.) is an important urban vector of several human 51

arboviruses including dengue, yellow fever, Zika and chikungunya viruses (Gubler 52

2008, Fauci et al. 2016). As there is no commercially available vaccine to prevent 53

dengue, Zika and chikungunya, vector control remains the primary method to prevent 54

outbreaks of these diseases. Surveillance for Aedes vectors of dengue has historically 55

concentrated on immature stages; however, immature monitoring generally fails to 56

correlate well with dengue risk (Focks et al. 2000, Bowman et al. 2014). This has 57

shifted the emphasis to improving adult Aedes monitoring (Achee et al. 2015). 58

Measurement of adult populations allows for direct monitoring of the impact of vector 59

control on epidemiologically significant populations of adult females, as well as the 60

capacity to test captured females for arboviruses and the presence of insecticide 61

resistance alleles. Furthermore, the use of population modification interventions 62

involving the release of Ae. aegypti infected with the bacterium Wolbachia require 63

careful measurement of the infection frequency in adult mosquito populations (Moreira 64

et al. 2009, Hoffmann et al. 2011, Walker et al. 2011). Other interventions also require 65

monitoring of adult populations, for example, these include efficacy assessments of 66

insecticide-treated materials (Kroeger et al. 2006, Lenhart et al. 2008, Andrade & 67

Cabrini 2010), the release of sterile (Alphey et al. 2010, Whyard et al. 2015) or 68

genetically modified insects (Lacroix et al. 2012), and the dispersion of spatial 69

repellents (Lloyd et al. 2013, Salazar et al. 2013). 70

A range of traps has been deployed for monitoring Ae. aegypti populations by 71

sampling eggs (ovitrap), host-seeking females (BG-Sentinel and BG-Mosquitito) or 72

gravid mosquitoes (MosquiTRAP, Sticky trap, double sticky ovitrap and CDC autocidal 73

gravid ovitrap (CDC-AGO). The ovitrap was developed in the 1960s (Fay & Eliason 74

Page 4 of 28



5

1966) and is still being used for detection or as a monitoring tool, especially when 75

vector populations are low. Ovitraps typically consist of a black container holding up to 76

1000 mL of water, containing a wooden paddle or cloth strip upon which gravid females 77

oviposit. However, oviposition substrates must be collected, incubated in the laboratory, 78

eggs then hatched and larvae reared for identification. Therefore, it is a laborious 79

methodology that provides information about the vector population with at least one or 80

two weeks delay (Reiter et al. 1991, Ritchie et al. 2003, Chadee & Ritchie 2010). As an 81

alternative to ovitraps, sticky ovitraps were developed to directly capture gravid females 82

and avoid the delays and logistics associated with egg hatching (Ritchie et al. 2003, 83

Eiras & Resende 2009, Barrera et al. 2014). The sticky ovitraps of Chadee and Ritchie 84

(2010) and Barrera et al. (2014) employ a wet glue (Atlantic Paste and Glue UVR-32), 85

while the MosquiTRAP of Fávaro et al. (2006) and Eiras & Resende (2009) use a dry 86

glue sticky card. The wet glues tend to capture a higher proportion of visiting female 87

Ae. aegypti than dry glues (S.A.R, unpublished data), but the glue adheres to skin when 88

touched. This messiness is loathed by field workers (Azil et al. 2014) and can damage 89

captured insects (Eiras et al. 2014, Ritchie et al. 2014). The BG-Sentinel® (Krockel et al 90

2006) and BG-Mosquito® (Hapairai et al. 2013) use lures and powered fans to capture 91

adult mosquitoes, requiring main power or batteries. They are relatively expensive and 92

may not be acceptable in areas without main power or because of the additional costs in 93

the electricity bill. Moreover, if a power failure occurs, it can trigger a failure of the 94

collections (Degener et al. 2013). 95

The Gravid Aedes Trap (GAT) was developed as an inexpensive, passive trap 96

that did not employ adhesives to capture gravid mosquitoes including Ae. aegypti (Eiras 97

et al. 2014). Upon entering the GAT, insects are killed by an insecticide surface spray in 98

about 15-30 min. The standard killing agent used in the GAT is a pyrethroid surface 99

Page 5 of 28



6

spray (Mortein Outdoor Barrier Surface Spray®; Imiprothrin 0.3 g/Kg, 0.6 g/Kg 100

Deltamethrin) that is applied to the bottom screen and the inner wall of the translucent 101

top of the GAT (Eiras et al. 2014). In both semi-field cage and field studies, the GAT 102

captured significantly more female Ae. aegypti than the MosquiTRAP and the double 103

sticky ovitrap (Chadee and Ritchie 2010), and, importantly, captured mosquitoes can be 104

processed for Wolbachia infection and dengue virus with no significant loss of 105

sensitivity (Ritchie et al. 2014). A commercial version of the GAT, the BG-GAT (order 106

Nr. 700, Biogents AG, Regensburg, Germany) was developed in 2014. 107

Although the surface spray has been highly efficient (Ritchie at al. 2014), 108

canned surface sprays may be unavailable, unacceptable to users, or ineffective against 109

pyrethroid resistant mosquitoes. Several alternative methods to capture adult 110

mosquitoes in the GAT are available. Long-lasting insecticide nets (LLINs) are 111

inexpensive and commonly available. Light oils could wet the wings of insects, making 112

flight and escape from the GAT difficult. Oil is also inexpensive, commonly available 113

and could be effective against insecticide resistant mosquitoes. Sticky cards are not 114

messy and have been widely employed to trap insects such as fruitflies (Heath et al. 115

1997) and even Aedes (Fávaro et al. 2006, Eiras & Resende 2009). Thus, we 116

investigated the use of alternative “killing” agents for use in the GAT, including non-117

insecticidal methods to develop an environmental friendly GAT. 118

Material and Methods 119

Aedes aegypti colony 120

Mosquitoes used in this study were from an established colony of wMel infected 121

Ae. aegypti sourced from Cairns (QLD, Australia) that is periodically supplemented 122

with wild collections to maintain genetic vigor. Mosquito larvae were reared on fish 123

food powder (TetraMin Tropical Flakes Fish Food, Tetra, Melle, Germany). Adults 124

Page 6 of 28



7

were feed on 50% honey solution and were blood-fed 3x week using human volunteers 125

(Human ethics approval from James Cook University H3555). Non-blood fed females 126

(nulliparous) of 5-15 days-old were used in laboratory experiments to measure the rate 127

of escape or the knockdown effect of the insecticides. These mosquitoes were more 128

active within the trap, therefore more likely to escape as they spend less time resting on 129

insecticide-treated surfaces (Eiras et al. 2014). Under semi-field conditions, only gravid 130

(5-6 d post blood-feeding) females were tested. Gravid females were transferred into a 131

clear plastic container (1 L) covered with a white mesh cloth (0.5 mm) with a sponge 132

(3.0 x 4.0 cm) soaked with honey solution (50%) provided as a sugar source. 133

Gravid Aedes Trap (GAT) and Bioassay Protocol 134

The GAT (Eiras et al. 2014) consists of a 10 L black bucket base, a translucent 135

top chamber, a black screen and a black plastic entrance funnel. The translucent 136

chamber consists of a circular plastic container, inverted and snugly inserted into the 137

black base. A black nylon mesh was placed between the translucent chamber and the 138

black base, separating both compartments, to prevent mosquito oviposition and retain 139

captured mosquitoes. The black entrance funnel (diameter 12 cm) was inserted on the 140

top of the translucent chamber and extended 6.5 cm into the GAT top (Eiras et al. 141

2014). Hay infusion of 7-15 d old prepared by adding 5 pellets (~2.5 g) of alfalfa in 3 L 142

of water was placed in the black bucket base as oviposition attractant. Later trials 143

(canola oil, adhesives) were conducted using the commercially available BG-GAT 144

(similar dimensions). 145

We used a modification of the standard “bench top” assay described by Eiras et 146

al. (2014) to measure the efficacy of the various insecticide and insecticide-free GAT 147

treatments. Briefly, a GAT containing water with the translucent top treated with the 148

killing agent was set on a laboratory bench. A cohort of ten 2-10 day old nulliparous 149

Page 7 of 28



8

female Ae. aegypti were carefully blown into the GAT head using a mouth aspirator. 150

Mosquitoes escaping from the entry funnel were captured in a 500 ml clear plastic cup 151

containing a strip of glue (Atlantic Paste and Glue UVR-32) inverted on top of the entry 152

funnel. Counts of dead or captured mosquitoes within the GAT head were made after 153

24 hr and, in the case of slow killing materials such as glue and oil, 48 hr to determine 154

the mean knockdown (KD) percent of each product. During all bioassays untreated 155

GATs served as negative controls and GATs treated with surface spray (Mortein 156

Outdoor Barrier Surface Spray®, imiprothrin 0.3 g/kg and 0.6 g/kg deltamethrin, Reckitt 157

Benckiser Pty. Ltd., West Ryde, New South Wales, Australia) served as positive 158

controls. The surface spray was applied to the inner wall of the translucent chamber and 159

to the black screen at least 24 hr before start the trial (Eiras et al. 2014, Ritchie et al. 160

2014). 161

Efficacy of LLINs to capture mosquitoes in the GAT 162

Several LLINs and net configurations were tested to determine which 163

combination provided the highest KD percentage of captured female Ae. aegypti in the 164

GAT. Initially, we measured 24 hr KD in a GAT containing LLINs treated with either 165

alphacypermethrin (4.8%) (supplied with the GAT, Biogents.com), deltamethrin (1.8 166

g/kg, Bestnet Netprotect®), or 2% permethrin and 1% piperonyl butoxide (Olyset 167

Plus®). A 25x25 cm square piece of each LLIN was placed loosely on the bottom mesh 168

of the GAT head in a nested configuration (nested bottom, Fig. 1a). We then assessed 169

several additional configurations using the Olyset Plus® LLIN, such as covering the 170

inner wall of the GAT, fitted to the top of the GAT and molded around the entry funnel 171

(22 cm diameter hole), fitted to the top and placed atop the bottom screen, hung 172

between one side of the entry funnel and on the bottom, and hung on either side of the 173

Page 8 of 28



9

entry funnel. Six replicates were conducted for each treatment with surface spray treated 174

GATs serving as positive controls. 175

Efficacy of vapor-active metofluthrin in the GAT 176

An early paper-based formulation of metofluthrin (Mortein Active Air®) was 177

shown to have fast KD of female Ae. aegypti in the GAT (Eiras et al. 2014). We 178

measured 24 hr KD percentage using a new longer lasting polyethylene mesh 179

formulation (SumiOne®, 212 mg metofluthrin impregnated sheet, Sumitomo Chemical 180

Australia Pty Ltd) (Ritchie & Devine 2013). A single piece of SumiOne® cut to either 181

1.0 cm2 or 2.5 cm2 in size was placed on the upper inner wall of GAT top. Mean escape 182

and KD percentages were assessed after 90 min and 24 hr. 183

Efficacy of dry stick card and canola oil in the GAT 184

A strip of dry sticky card (14 cm long by 7 cm and 3.5 cm wide on the bottom 185

and top, respectively, Fig. 1b) was attached between the entry funnel and the inner wall 186

of the translucent top to intercept mosquitoes flying between the funnel and trap wall. 187

The three dry glues tested included a yellow fly glue strip (David Grays Trappit Insect 188

Garden Trap®) and two sticky cards used in the MosquiTRAP (Gama et al. 2007, A. E. 189

unpublished data) that were applied to a brown plastic of the same dimension as the 190

Trappit sticky cards. The canola oil treatment consisted of an aerosolized canola oil 191

spray (Coles® Canola Oil Cooking Spray) that was lightly applied to the mesh bottom 192

and inner wall of the GAT top then spread into a light film using a paper towel. Both 193

24 and 48 hr KD of cohorts of 10 Ae. aegypti were recorded. The KD and escape results 194

of the glue and oil treatments were assessed against GATs treated with either surface 195

spray or those containing bed net (alphacypermethrin) that served as positive controls. 196

Impact of dry sticky card and canola residue on downstream molecular processing 197

Page 9 of 28



10

To assess the potential impact of dry glue or canola oil residue on downstream 198

molecular processing we submitted a subsample of 10 male and female Ae. aegypti for 199

Wolbachia detection by qPCR that had been exposed to either Trappit dry sticky panels 200

or canola oil for 48 hr and then held in a GAT for 1 week. At the end of the 1 week 201

holding period the specimens were preserved in 80% ethanol and submitted during 202

routine qPCR monitoring of Wolbachia infection frequency in wMel Ae. aegypti 203

following standard protocols (Lee et al. 2012). 204

Efficacy of insecticide and insecticide-free agents in the field 205

A series of Latin square design trials (Table 1) were conducted to compare 206

capture of female Ae. aegypti in GATs using insecticide (surface spray, LLIN, 207

metofluthrin) and insecticide-free alternative KD agents (sticky cards and canola oil) in 208

the field. The field trials were conducted at suburbs of Parramatta Park and Cairns 209

North, in Cairns Queensland, Australia that historically have high populations of Ae. 210

aegypti and dengue transmission (Ritchie et al. 2014). For each Latin square, all GATs 211

were set in shaded areas protected from rain at individual residences and each GAT was 212

baited with a hay infusion consisting of 3 g of hay to 3 L water at the beginning of each 213

Latin square. 214

Statistical Analysis 215

Differences in KD and escape percentage among the various ‘alternative’ GAT 216

treatments were assessed by analysis of variance (ANOVA) followed by Tukey HSD 217

post-hoc analysis, both with 5% significance. The mean weekly number of female Ae. 218

aegypti collected per trap during field Latin square trials was analysed by ANOVA 219

followed by Tukey HSD post-hoc analysis. The explanatory variables were: (a) 220

residential address, (b) week, and (d) treatment. The R program version 3.1.0 221

(http://www.R-project.org) was used to perform all the statistical analysis and the 222

Page 10 of 28



11

graphics produced using GraphPad Prism ver. 5.0 (GraphPad Software, San Diego 223

California USA. 224

Results 225

Use of LLINs to capture mosquitoes in the GAT 226

No significant difference in KD (F7,80=1.7, P=0.12) or escape (F5,35=1.8, 227

P=0.14) was observed among the different LLIN treatments and configurations after 90 228

min or 24 hr of exposure (Table 2). Overall, KD percentages ranged from 78.7±15.4 to 229

97.2±6.8% and escape percentages ranged from 3.1±1 to 8.7±4.2% after 24 hr. The 230

nested bottom LLIN configuration generally produced the greatest KD percentages 231

compared to the other configurations tested (Table 2) while also being the easiest 232

configuration to set within the GAT. 233

Efficacy of vapor-active metofluthrin in the GAT 234

GATs containing a 1.0 cm2 strip of metofluthrin produced significantly lower 235

KD percentages (F3,6=12.27, P=0.043) and significantly higher escape percentages 236

(F3,6=8.18, P=0.047) compared to GATs set with a 2.5 cm2 strip of Metofluthrin® 237

(Table 2). Overall, the mean 90 min KD percent in control GATs and GATs set with the 238

2.5 cm2 strip of Metofluthrin® was 93.1±8.3% and 89.1±8.2%, respectively, whereas the 239

mean KD percent in GATs set with the 1 cm2 Metofluthrin® strip was 77.7±15.3%. 240

Additionally, mean escape percent was 6.9±3.1% to 10.9±4.1% in the control and 2.5 241

cm2 Metofluthrin® treated GATs, respectively, whereas it was 24.8±9.7% in the 1 cm2 242

Metofluthrin® treated GATs. 243

Efficacy of dry sticky cards and canola oil in the GAT 244

No significant differences in KD (F2,15=0.90, P=0.43) or escape (F2,15=1.23, 245

P=0.32) were observed among the sticky cards and canola oil treatments compared to 246

control (surface spray) GATs (Fig. 2, Table 2). KD percentages in sticky card and 247

Page 11 of 28



12

canola oil treatments ranged from 70±7.7% to 90.0±3.7% and 81±8.8% to 91.3±3.3% 248

after 24 and 48 hr, respectively. Mean 48 hr escape percentages ranged from 4.7±7.2% 249

to 25±7.2%. Overall, the canola oil treatment experienced the lowest 24 hr KD 250

percentage (70±7.7%) while the MosquiTRAP sticky card had the highest 24 hr KD 251

percentage (87.8±10.5%) among the insecticide-free treatments, which improved to 252

81±8.7% and 91.8±9.5%, respectively, after 48 hr. 253

Impact of dry sticky card and canola residue on downstream molecular processing 254

Each alternative agent was found to have no inhibitory effects on downstream 255

molecular processing as all samples were positively identified as Ae. aegypti and all 256

tested positive for the presence of Wolbachia. 257

Efficacy of insecticide and insecticide-free agents in the field 258

No significant difference (F2,93=0.02, P=0.98) in the number of Ae. aegypti 259

females captured across the different insecticide-based treatments was observed during 260

the first field trial (Fig. 3 a). Overall, the lowest percentage of traps positive for female 261

Ae. aegypti were the metofluthrin treated GATs (50.0%), whereas the highest 262

percentage of traps positive for Ae. aegypti were the control GATs (surface spray, 263

82.1%), while 71.4% of bed net treated GATs were positive for Ae. aegypti. Similarly, 264

no significant differences were observed among the different insecticide and insecticide-265

free treatments during the second and third field trials (F2, 93=0.02, P=0.98 and F2, 266

51=1.02, P=0.37, respectively). The mean number of female Ae. aegypti collected per 267

week across all GAT treatments ranged from 2±1.03 to 2.09±0.31 and 1.17±0.43 to 268

1.87±0.38 during the second and third field trials, respectively (Fig. 3 b, c). Among the 269

insecticide-free treatments, the canola oil had the lowest mean percentage of traps 270

positive for Ae. aegypti (66.7%), whereas GATs treated with sticky card (Trappit) had a 271

higher percentage of traps positive for Ae. aegypti than the LLIN and surface spray 272

Page 12 of 28



13

treated GATs during the second and third field trials, respectively (87.5% and 88.9% vs 273

68.8% and 83.3%). 274

Discussion 275

The GAT is an effective and inexpensive surveillance tool for adult Ae. aegypti 276

and, potentially, other container-inhabiting species such as Aedes albopictus (Skuse) 277

(Ritchie et al. 2014). The GAT can also be used to detect viruses in captured 278

mosquitoes (Ritchie et al. 2014), and may be particularly useful for monitoring for Zika 279

virus (ZIKV); because most human infections are subclincal, mosquito infections will 280

be a key to monitoring ZIKV presence (Loos et al. 2014, Chen and Hamer 2016). In 281

this study, we highlight the successful incorporation of several ‘alternative’ insecticide 282

and insecticide-free killing agents in the GAT to capture gravid Ae. aegypti under 283

laboratory and field conditions. The development of insecticide-free alternatives is 284

particularly important due to the continued emergence of insecticide resistance in Ae. 285

aegypti populations across the world, especially in developing countries in South 286

America and Southeast Asia (Vontas et al. 2012). The use of sticky cards and other 287

glue-based agents have been incorporated successfully in a variety of traps used to 288

monitor Ae. aegypti populations, such as the double sticky ovitrap (Chadee and Ritchie 289

2010), CDC-AGO (Barrera et al. 2014), and the MosquiTRAP (Eiras & Resende, 2009). 290

However, the primary advantages of the “dry” Trappit and MosquiTRAP sticky cards, 291

as well as the use of canola oil, over “wet” glues, such as those used in the double sticky 292

ovitrap and CDC-AGO, are the non-mess handling that allows for easy removal of 293

mosquitoes. Neither of the dry glues tested nor canola oil inhibited downstream 294

molecular processing for species identification and the detection of Wolbachia 295

infection. Although the KD percentages of the insecticide-free alternatives were 296

generally lower than the insecticide treatments in a laboratory setting, no significant 297

Page 13 of 28



14

difference in efficacy was observed among the various ‘alternative’ agents under field 298

conditions. This is likely due to the low escape rate of Ae. aegypti from the GATs, 299

which allows them to be exposed to the alternatives for longer periods resulting in 300

similar capture rates to insecticide-based agents. The use of such environmentally 301

friendly alternatives also has the added benefit of circumventing resistance to 302

insecticide use due to environmental and public health concerns. 303

Although the originally recommend use of pyrethroid-based surface sprays in 304

the GAT is highly effective (Ritchie at al. 2014), canned surface sprays may be 305

unavailable in many countries, unacceptable to users, or ineffective against pyrethroid 306

resistant mosquitoes. Long-lasting insecticide nets (LLINs) are an attractive alternative 307

as they are commonly available and come treated with a wide range of active 308

ingredients, as well as the addition of synergist compounds to increase efficacy. The use 309

of LLINs in the GAT decrease exposure of field workers to insecticides since GATs 310

must be retreated monthly when using surface spray, whereas many LLINs maintain 311

their efficacy for up to two or more years (WHO 2013, Odhiambo et al. 2013). Because 312

of these advantages, we evaluated the efficacy of two single active compound LLINs 313

(NetProtect, deltamethrin 1.8 g/kg; GAT bed net, 4.8% alphacypermethrin) and the dual 314

compound Olyset Plus® (2% permethrin, 1% piperonyl butoxide). The dual compound 315

OlysetPlus® has been demonstrated to be more effective than single compound products 316

against resistant mosquitoes due to the presence of the synergist piperonyl-butoxide, 317

which enhances the potency of certain insecticides, including synthetic pyrethroids, by 318

increasing its absorption by target insects (Fakoorziba et al. 2009, Pennetier et al. 2014). 319

During our laboratory and field studies both of the single active compound LLINs and 320

the OlysetPlus® LLIN performed equally well. It should be noted that these results were 321

obtained against susceptible laboratory colonies and against field populations with no 322

Page 14 of 28



15

history of resistance to pyrethroids. Based on previous reports, it is likely that the 323

OlysetPlus® LLIN would have outperformed the two single compound LLINs if tested 324

against populations with varying levels of pyrethroid resistance (Pennetier et al. 2014). 325

However, in areas where pyrethroid resistance is not an issue, the less expensive single 326

compound LLINs will provide excellent capture rates. With regards to the different 327

LLIN trap configurations tested (e.g. funnel, sides, nested bottom), it is our 328

recommendation that the nested bottom configuration be used as it is the simplest to set 329

and allows for persistent contact of the LLIN to captured mosquitoes as most are 330

trapped between the LLIN and the trap mesh. 331

In addition to the different LLINs tested, the metofluthrin-based product tested 332

provided comparable KD percentages as the LLINs and surface spray. Although 333

metofluthrin has been labeled as a spatial repellent for mosquitoes (Lloyd et al. 2013) 334

and has been shown to reduce captures of mosquitoes (Lloyd et al. 2013, Dame et al. 335

2014), the number of Ae. aegypti females captured in the field did not differ among 336

metofluthrin, LLIN, and surface spray treated GATs. However, we did observe a 337

substantial reduction in the number of traps positive for Ae. aegypti between 338

metofluthrin (50%) and surface spray (82%) treated GATs. These results suggest that 339

the high spatial repellency or confusion induced by metofluthrin, although partial and 340

nonspecific (Xue et al. 2012), may have discouraged gravid females from entering the 341

GATs. While lower catches may have been the result of site-specific characteristics 342

(i.e. wind speed, ventilation), this could still compromise metofluthrin’s application for 343

use in the GAT. In addition, metofluthrin has no long term durability, remaining active 344

for up to 20 days (Ritchie & Devine 2013), whereas the Mortein® surface spray tested 345

can last up to eight weeks, (Ritchie et al. 2014) and LLIN potentially much longer. 346

Conclusions 347

Page 15 of 28



16

The identification of ‘alternative’ insecticide and insecticide-free killing agents 348

is essential to ensure the GAT remains an effective surveillance device against resistant 349

populations of Ae. aegypti (Flores et al. 2013, Maciel-de-Freitas et al. 2014). Moreover, 350

although the originally recommended use of residual insecticide surface sprays is 351

effective, these sprays must be reapplied monthly and may be unavailable in certain 352

locations. In contrast, we demonstrate that widely available LLINs, which remain active 353

for upwards of two years, provide excellent Ae. aegypti capture rates. In addition to the 354

products tested in the current study, there are many types of LLINs available, 355

particularly those containing synergist compounds, that help to maintain their efficacy 356

even against resistant populations. In contrast, we do not recommend the use of 357

metofluthrin due to its repellency characteristics that may negatively affect the 358

attractiveness of the GAT to gravid females. Perhaps most importantly, we demonstrate 359

that simple insecticide-free alternatives, including sticky cards and canola oil sprays, 360

performed equally well compared to the insecticide-based products tested under field 361

conditions. The use of inexpensive, widely available, and environmentally friendly 362

insecticide-free agents such of those described in this study will be particularly 363

attractive in areas with pyrethroid resistant populations and areas where there is 364

substantial resistance to insecticide use due to environmental and human health 365

concerns. 366

Acknowledgments 367

We thank Doreen Weatherby for providing the BestNet NetProtect LLINs, and John 368

Lucas and Garry Webb of Sumitomo for providing the Olyset Plus LLIN. We also 369

thanks to Biogents (Germany) for providing the BG-GAT prototype for testing. AEE 370

thanks CAPES and CNPq for providing funding. 371

372

Page 16 of 28



17

References 373

Achee, N. L., F. Gould, T. A. Perkins, R. C. Reiner, A. C. Morrison, S. A. Ritchie, 374

D. J. Gubler, R. Teyssou, and T.W. Scott. 2015. A critical assessment of vector 375

control for dengue prevention. PLoS Negl Trop Dis 9(5): e0003655. 376

doi:10.1371/journal.pntd.0003655. 377

Alphey, L., M. Benedict, R. Bellini, G. G. Clark, D. A. Dame, M. W. Service, et al. 378

2010. Sterile-insect methods for control of mosquito-borne diseases: an analysis. 379

Vector Borne Zoonotic Dis. 10: 295-311. 380

Andrade C.F.S., and I. Cabrini. 2010. Comparative studies on Aedes aegypti and 381

Aedes albopictus adult females trespassing commercial nets. J Am Mosq Cont 382

Assoc. 26(1): 112-115. 383

Barrera, R., M. Amador, V. Acevedo, B. Caban, G. Felix, and A. J. Mackay. 2014. 384

Use of the CDC Autocidal Gravid Ovitrap to Control and Prevent Outbreaks of 385

Aedesaegypti (Diptera: Culicidae). J Med Entomol. 51: 145-451. 386

Bowman LR, Runge-Ranzinger S, McCall PJ. 2014. Assessing the relationship 387

between vector indices and dengue transmission: A systematic review of the 388

evidence. PLoS Negl Trop Dis. 2014; 8(5). doi: 10.1371/journal.pntd.0002848 389

Chadee, D. D., and S.A. Ritchie. 2010. Efficacy of sticky and standard ovitraps for 390

Aedes aegypti in Trinidad, West Indies. J Vector Ecol. 35: 395-400. 391

Chen, L. H., and D. H. Hamer. 2016. Zika virus: Rapid spread in the Western 392

Hemisphere. Ann Intern Med. doi:10.7326/M16-0150 393

Dame, D.A., M. V. Meisch, C. N. Lewis, D. L. Kline, and G.G Clark. 2014. Field 394

evaluation of four spatial repellent devices against Arkansas rice-land mosquitoes. 395

J Am Mosq Control Assoc. 30:31-36. 396

Page 17 of 28



18

Degener, C.M., A. E. Eiras, T. M. F. Azara, R. A. Roque, S. Rösner, C. T. Codeço, 397

A. A. Nobre, et al. 2014. Evaluation of the effectiveness of mass trapping with 398

BG-sentinel traps for dengue vector control: a cluster randomized controlled trial 399

in Manaus, Brazil. J Med Entomol 51(2): 408-420. 400

Eiras, A.E., and M.C. Resende. 2009. Preliminary evaluation of the "Dengue-MI" 401

technology for Aedes aegypti monitoring and control. Cadernos de Saúde Pública. 402

25: S45-S58. 403

Eiras, A.E., T. S. Buhagiar, and S.A. Ritchie. 2014. Development of the Gravid 404

Aedes Trap for the capture of adult female container–exploiting mosquitoes 405

(Diptera: Culicidae). J Med Entomol. 51(1): 200-209. 406

Fakoorziba, M.R., F. Eghbal, and V.A. Vijayan. 2009. Synergist efficacy of 407

piperonyl butoxide with deltamethrin as pyrethroid insecticide on Culex 408

tritaeniorhynchus (Diptera: Culicidae) and other mosquitoes species. Environ 409

Toxicol. 24:19-24. 410

Fauci, A. S., and D.M. Morens. . 2016. Zika virus in the Americas-Yet another 411

arbovirus threat. N Engl J Med. 374:601-604. doi: 10.1056/NEJMp1600297 412

Fávaro, E.A., M. R. Dibo, A. Mondini, A. C. Ferreira, A. C. Barbosa, A. E. Eiras, 413

E. M. Barata, and F. Chiaravalloti-Neto. 2006. Physiological state of Aedes 414

(Stegomyia) aegypti mosquitoes captured with MosquiTRAPs™ in Mirassol, São 415

Paulo, Brazil. J Vector Ecol 31(2): 285-291. 416

Flores, A. E., G. Ponce, B. G. Silva, S. M. Gutierrez, C. Bobadilla, B. Lopez, R. 417

Mercado , and W. C. Black. 2013. Wide spread cross resistance to pyrethroids in 418

Aedes aegypti (Diptera: Culicidae) from Veracruz state Mexico. J Econ Entomol 419

Focks DA, Brenner RJ, Hayes J, Daniels E. 2000. Transmission thresholds for 420

dengue in terms of Aedes aegypti pupae per person with discussion of their utility 421

Page 18 of 28



19

in source reduction efforts. Am J Trop Med Hyg. 2000; 62(1): 11–18. PMID: 422

10761719106:959-969. 423

Gama, R. A., E. M. Silva, I. M. Silva, M. C. Resende, and A. E. Eiras. 2007. 424

Evaluation of the sticky MosquiTRAP™ for detecting Aedes (Stegomyia) aegypti 425

(L.) (Diptera: Culicidae) during the dry season in Belo Horizonte, Minas Gerais, 426

Brazil. Neotropical Entomol. 36(2): 294-302. 427

Gubler, D. J.. 2008.The global threat of emergent/reemergent vector-borne diseases. In 428

Vector-Borne Diseases: understanding the environmental, human health, and 429

ecological connections, The National Academies Press, Institute of Medicine, 430

Washington, D.C., p. 43-64. 431

Hapairai, L. K., H. Joseph, M. A. Cheong Sang, W. Melrose, S. A. Ritchie, T. R. 432

Burkot, S. P. Sinkins, and B. C. Bossin. 2013. Field evaluation of selected traps 433

and lures for monitoring the filarial and arbovirus vector, Aedes polynesiensis 434

(Diptera: Culicidae), in French Polynesia. J Med Entomol 50(4): 731-739. 435

Heath, R. R., N. D. Epsky, B. D. Dueben, J. Rizzo, and F. Jeronimo. 1997. Adding 436

methyl-substituted ammonia derivatives to a food-based synthetic attractant on 437

capture of the Mediterranean and Mexican fruit flies (Diptera: Tephritidae). J 438

Econ Entomol 90: 1584-1589. 439

Hoffmann, A. A., B. L. Montgomery, J. Popovici, I. Iturbe-Ormaetxe, P. H. 440

Johnson, F. Muzzi, M. Greenfield, et al. 2011. Successful establishment of 441

Wolbachia in Aedes populations to suppress dengue transmission. Nature. 476 442

(7361): 454-457. 443

Kroeger, A., A. Lenhart, M. Ochoa, E. Villegas, M. Levy, N. Alexander, and P. J. 444

McCall. 2006. Effective control of dengue vectors with curtains and water 445

Page 19 of 28



20

container covers treated with insecticide in Mexico and Venezuela: cluster 446

randomised trials. BMJ. 332(7552): 1247-1252. 447

Kroeckel, U., A. Rose, A. E. Eiras, M. Geier. 2006. New tools for surveillance of 448

adult yellow fever mosquitoes: comparison of trap catches with human landing 449

rates in an urban environment. J Am Mosq Control Assoc 22(2): 229-238. 450

Lacroix, R., A. R. McKemey, N. Raduan, W. L. Kwee Wee, W. Hong Min, T. Guat 451

Ney, A. A. Siti Rahidah, et al. 2012. Open field release of genetically engineered 452

sterile male Aedes aegypti in Malaysia. PLoS One: e42771. 453

Lee, S. F., V. L. White, A. R. Weeks, A. A. Hoffmann, and N. M. Endersb. 2012. 454

High-throughput PCR assays to monitor Wolbachia infection in the dengue 455

mosquito (Aedes aegypti) and Drosophila simulans. App Environ Microbio 456

78(13): 4740-4743. 457

Lenhart, A., N. Orelus, R. Maskill, N. Alexander, T. Streit, and P. J. McCall. 2008. 458

Insecticide‐treated bednets to control dengue vectors: preliminary evidence from a 459

controlled trial in Haiti. Trop Med Int Health. 13(1): 56-67. 460

Lloyd, A. M., M. Farooq, J. W. Diclaro, D. L. Kline, and A. S. Estep. 2013. Field 461

evaluation of commercial off-the shelf spatial repellents against the Asian Tiger 462

Mosquito, Aedes albopictus (Skuse), and the potential for use during deployment. 463

US Army Med Dept J: 80-86. 464

Loos, S., H. P. Mallet, I. Leparc Goffart, V. Gauthier, T. Cardoso, and M. Herida. 465

2014. Current Zika vius epidemiology and recent epidemics. Médecine et 466

Maladies Infectieuses. 44 (7): 302-307. 467

Maciel-de-Freitas, R., F. C. Avendanho, R. Santos, S. Gabriel, S. C. Araújo, J. B. 468

P. Lima, A. J. Martins, G. E. Coelho, and D. Valle. 2014. Undesirable 469

Page 20 of 28



21

consequences of insecticide resistance following Aedes aegypti control activities 470

due to a dengue outbreak. PLoS One. 9: e92424. 471

Moreira, L. A., I. Iturbe-Ormaetxe, J. A. Jeffery, G. Lu, A. T. Pyke, L. M. Hedges, 472

B. C. Rocha, et al. 2009. A Wolbachia symbiont in Aedes aegypti limits infection 473

with dengue, Chikungunya, and Plasmodium. Cell 139(7): 1268-1278. 474

Odhiambo, M. T. O., O. Skovmand, J. M. Vulule, and E. D. Kokwaro. 2013. 475

Polyethylene-based long lasting treated bed net Netprotect on Anopheles 476

mosquitoes, malaria incidence, and net longivity in Western Kenya. J Trop Med 477

2013: 1-10. 478

Pennetier, C., A. Bouraima, F. Chandre, M. Piameu, J. Etang, M. Rossignol, I. 479

Sidick, B. Zogo, M. I. Lacroix, R. Yadav, O. Pigeon, and V. Corbel. 2014. 480

Efficacy of Olyset Plus, a new long-lasting insecticidal net incorporating 481

permethrin and piperonil-butoxide against multi-resistant malaria vectors. PLoS 482

One 8: e 75134. 483

Ritchie, S. A., S. Long, A. Hart, C. E. Webb, and R. C. Russell. 2003. An adulticidal 484

sticky ovitrap for sampling container-breeding mosquitoes. J Am Mosq Cont 485

Assoc 19(3): 235-242. 486

Ritchie, S. A., and G. J. Devine. 2013. Confusion, knock-down and kill of Aedes 487

aegypti using metofluthrin in domestic settings: a powerful tool to prevent dengue 488

transmission. Parasites & Vectors 6: 262-280. doi:10.1186/1756-3305-6-262. 489

Ritchie, S. A., T. S. Buhagiar, M. Townsend, Hoffmann A, Van Den Hurk AF, 490

McMahon JL, Eiras AE. 2014. Field validation of the Gravid Aedes Trap (GAT) 491

for collection of Aedes aegypti (Diptera: Culicidae). J Med Entomol 51:210-219. 492

Salazar, F. V., N. L. Achee, J. P. Grieco, A. Prabaripai, T. A. Ojo, L. Eisen, C. 493

Dureza, S. Polsomboon, and T. Chareonviriyaphap. 2013. Effect of Aedes 494

Page 21 of 28



22

aegypti exposure to spatial repellent chemicals on BG-Sentinel™ trap catches. 495

Parasites & Vectors 6: 145. 496

Vontas, J.E., Kioulos, N., Pavlidi, E., Morou, A., Torre, D., and H. Ranson H. 2012. 497

Insecticide resistance in the major dengue vectors Aedes albopictus and Aedes 498

aegypti. Pesticide Biochem Physiology 104(2): 126-131. 499

Walker, T., P. H. Johnson, L. A. Moreira, I. Iturbe-Ormaetxe, F. D. Frentiu, C. J. 500

McMeniman, Y. San Leong, et al. 2011. The wMel Wolbachia strain blocks 501

dengue and invades caged Aedes aegypti populations. Nature 476: 450-453. 502

WHO 2013. World Malaria Report 2013, World Health Organization, Geneva 255pp. 503

Whyard, S., C. N. Erdelyan, A. L. Partridge, A. D. Singh, N. W. Beebe, and R. 504

Capina. 2015. Silencing the buzz: a new approach to population suppression of 505

mosquitoes by feeding larvae double-stranded RNAs. Parasites & Vectors 8(1): 506

96. doi:10.1186/s13071-015-0716-6. 507

Xue, R.D., W. A. Qualls, M. L. Smith, M. K. Gaines, J. H. Weaver, and M. 508

Debboun. 2012. Field evaluation of the Off! Clip-on Mosquito Repellent 509

(metofluthrin) against Aedes albopictus and Aedes taeniorhynchus (Diptera: 510

Culicidae) in northeastern Florida. J Med Entomol 49:652-655. 511

Page 22 of 28



23

Table 1. Summary of the killing agents assessed and number of replicates performed in 512

each field Latin square study. 513

Latin Square 1:

4 replicates

Latin Square 2:

3 replicates

Latin Square 3:

3 replicates

Mortein® Surface Spray Mortein® Surface Spray Mortein® Surface Spray

LLIN (Bestnet Netprotect®): Nested Bottom

LLIN (Bestnet Netprotect®): Nested Bottom

MosquiTrap Dry Stick Card

LLIN (Bestnet Netprotect®): Bottom and Side

MosquiTrap Dry Stick Card

Canola Oil

2.5 cm2 square of SumiOne® metofluthrin

Page 23 of 28



24

Table 2. Knockdown and capture rates of female Aedes aegypti in Gravid Aedes Traps 514

treated with alternative agents. SD represents standard deviation. 515

I. LLIN Brand Active ingredient Configuration Mean (SD) 24

hr KD %

BG-GAT Alphacypermethrin (4.8%)

Nested bottom 93.3 (6.7)

Bestnet Netprotect®

deltamethrin 1.8 g/kg

Nested bottom Male: 90 (10.9) Female: 97 (5.1)

Olyset Plus 2% permethrin 1% piperonyl butoxide

Nested bottom 92.2 (9.8)

Olyset Plus Side of top, bottom

94.4 (6.6)

Olyset Plus Side entry funnel, bottom

97.2 (6.8)

Olyset Plus Bottom 89.0 (6.0) Olyset Plus Nested bottom 92.2 (9.8) Olyset Plus 2 strips hung

between entry funnel and top

78.7 (15.4)

Metofluthrin SumiOne 10% metofluthrin 1 cm2 piece 77.7 (15.2) SumiOne 10% metofluthrin 2.5 cm2 piece 87.6 (6.6)

Sticky card Dry glue MosquiTRAP (24 hr)

Piece hung between entry funnel and GAT head

87.8 (10.5)

Dry glue MosquiTRAP (48 hr)

Piece hung between entry funnel and GAT head

91.8 (9.5)

Dry glue Trappit yellow sticky insect trap (24 hr)

Piece hung between funnel and GAT head

Female: 79 (7.1) Male: 81 (8.1)

Trappit yellow sticky insect trap (48 hr)

Piece hung between funnel and GAT head

Female: 90 (8.9) Male: 95 (8.4)

Oil Canola oil Coles Canola Oil Cooking Spray (48 hr)

Thin film of oil on inside of translucent head

48 hr KD Male: 96.7 (5.2) Female: 81 (8.7)

Page 24 of 28



25

Fig. 1. (A) Illustration of the “nested bottom” long-lasting insecticide-impregnated net 516

configuration and (B) positioning of the dry sticky cards when used in the Gravid Aedes 517

Trap. 518

Fig. 2. Percentage (mean ± SE) of Ae. aegypti females escaped and knocked down after 519

48 hr in GATs containing Mortein® surface spray, Olyset Plus LLIN, MosquiTRAP dry 520

sticky card, Trappit dry sticky card, or treated with canola oil under laboratory 521

conditions. The “a” and associated black bar represents no statistical difference (P-value 522

< 0.05, ANOVA, Tukey HSD post-hoc analysis) among the treatments. 523

Fig. 3. (A) Mean (±SE) number of Ae. aegypti captured per week in GATs treated with 524

standard surface spray, containing a strip of metofluthrin, and different LLIN 525

configurations. (B) Mean (±SE) number of Ae. aegypti captured per week in GATs 526

treated with dry sticky card compared to insecticide treatments (surface spray and 527

LLIN). (C) Mean (±SE) number of Ae. aegypti captured per week in GAT treated with 528

canola oil compared to surface spray and dry sticky cards. 529

530

Page 25 of 28



Fig. 1. (A) Illustration of the “nested bottom” long-lasting insecticide-impregnated net configuration and (B) positioning of the dry sticky cards when used in the Gravid Aedes Trap.

287x190mm (72 x 72 DPI)

Page 26 of 28



Percentage (mean ± SE) of Ae. aegypti females escaped and knocked down after 48 hr in GATs containing Mortein® surface spray, Olyset Plus LLIN, MosquiTRAP dry sticky card, Trappit dry sticky card, or treated with canola oil under laboratory conditions. The “a” and associated black bar represents no statistical

difference (P-value < 0.05, ANOVA, Tukey HSD post-hoc analysis) among the treatments. 108x68mm (300 x 300 DPI)

Page 27 of 28



Fig. 3. (A) Mean (±SE) number of Ae. aegypti captured per week in GATs treated with standard surface spray, containing a strip of metofluthrin, and different LLIN configurations. (B) Mean (±SE) number of Ae. aegypti captured per week in GATs treated with dry sticky card compared to insecticide treatments (surface

spray and LLIN). (C) Mean (±SE) number of Ae. aegypti captured per week in GAT treated with canola oil compared to surface spray and dry sticky cards.

94x238mm (300 x 300 DPI)

Page 28 of 28



Documents

Evaluation of alternative killing agents for Aedes … · Evaluation of alternative killing agents for Aedes aegypti (Diptera: Culicidae) in the Gravid Aedes Trap (GAT) Journal: Journal